Throughout this application various publications are referred to by number in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Some eukaryotic parasites, such as Plasmodium species that cause malaria, Leshmania species that cause leshmaniasis, Trypanasoma species that cause African sleeping sickness, Chagas disease and Toxoplasma gondii that causes toxoplasmosis, and Cryptosporidium which causes cryptosporidiosis, are purine auxotrophs, unable to perform de novo purine biosynthesis [3-7]. Because nucleobases and nucleosides are impermeable through phospholipid cell membranes, cells use Equilibrative Nucleoside Transporters (ENTs) and Concentrative Nucleoside Transporters (CNTs) to import and export purines and pyrimidines [1,2]. The parasites rely on purine import via ENTs, and possibly CNTs, to supply purines needed for DNA synthesis and other cellular processes. The imported purines are processed through the purine salvage pathway to generate the specific purines required by the cell.
Blocking purine import is likely to have inhibitory or cytotoxic effects on these parasites. For example, knockout of the Plasmodium falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1) results in parasites that are not viable during in vitro culture in growth media containing physiological purine concentrations found in normal human blood [8,9].
ENTs are also present in humans where they are responsible for clearance of nucleobases and nucleosides from the plasma and extracellular space to terminate purinergic signaling [10-13]. Human ENTs are about 18% sequence identical with PfENT1. It is reasonable to expect that species specific ENT inhibitors exist. Supporting this, human ENT inhibitors, dipyridamole, dilazep and p-nitrobenzylthioinosine, do not inhibit PfENT1. In addition, dipyridamole and dilazep are in clinical use as coronary artery vasodilators and antiplatelet agents. Thus, ENT inhibitors are useful therapeutic agents for modulating purinergic signaling pathways. Moreover, some cancer cells are unable to perform de novo purine biosynthesis and are reliant on purine uptake for DNA replication. ENT inhibitors can serve as anti-cancer drugs for this limited group of cancers.
The present invention discloses a novel high throughput assay and system for identifying ENT inhibitors.